Edulcorantes para bebidas aprobados en diferentes países

2.3.3.1 Probe Preparation

Whole mount in situ hybridisation was used to examine the expression of the genes of interest in day 6.5-7.5 mouse embryos.

2.3.3.2 Primer Design

Primers were designed to amplify the 3’ end of the gene of interest. The optimal length of the PCR product was 700-1000 base pairs. Highly conserved regions of the gene should be avoided so the probes do not hybridise with mRNA of other genes. The prevention of cross hybridisation can be ensured by using the Ensembl web site: http://www.ensembl.org/Mus_musculus/index.html and determining how many members are present in the superfamily. If there were many members in the superfamily a blast search was carried out

http://blast.ncbi.nlm.nih.gov against the mouse genome. If the overall identity of the two genes was over 85 % that area that showed possible cross hybridisation was excluded and also short stretches with over 95 % identity were also to be excluded. If the region could not be excluded a note was taken in case of cross hybridisation later on. Primers were designed by using vector NTi software or by eye. The sequence of each gene was also checked for abnormally high or low GC content. As cloning was carried out by TA cloning, primers were designed with an A or a G at the 5’ end as Taq polymerase is more likely to put an A on the 3’ end of the product. Adding an A at the 5’ end of the primer increases cloning efficiency (Peng et al., 2007).

Primers were ordered from Sigma and they were re-suspended in Tris-EDTA to give a 1 mM stock solution and were stored at -80 °C. Primers were then diluted 1:100 in DEPC treated water to give a 10 µM working solution, they are stored at -20 °C.

45 2.3.3.3 Cloning using PCR

The genes of interest were amplified using PCR. The PCR was carried out in a PTC-200 Peltier Thermal Cycler containing the following reaction mix;

MQ water 40.5 µl 10 x PCR buffer 5 µl dNTP mix (10 mM) 1 µl DNA template 1 µl Forward primer (10 µM) 1 µl Reverse primer 10 µM 1 µl FastStart Taq Polymerase 0.5 µl

The 10x PCR buffer provides a stable chemical environment for the Taq polymerase including Mg2+. The dNTP mix is deoxynucleotide triphosphates which were used as the building blocks for the new DNA. The samples were mixed and run at the following temperatures; 95 °C for 5 min (hot start), then 35 cycles of 94 °C 30 sec (denaturation), 60 °C 45 sec (annealing- primer dependant), 72 °C for 1 min (elongation). Then 72 °C for 10 min and then held at 4°C. Then 5 µl of the product was run on a 1 % agarose gel containing ethidium bromide with 1 µl gel loading buffer with a 100 bp ladder at 95 V until a good separation of the DNA bands was obtained.

2.3.3.4 Purification of the PCR Product

The PCR product had to be purified as Taq is difficult to inactivate and would fill in restricted ends, as primer dimers will compete for restriction enzymes and as nucleotides will interfere. Product purification was carried out using a Wizard® SV Gel and PCR Clean-Up System. The manufacturer’s instructions were followed see 2.3.2.4.1 above. The eluted DNA was stored at -20 °C until further use. 2.3.3.5 Ligation of DNA into the Vector

As Taq DNA polymerase adds a 3’ A to the end of the DNA strand (Zhou and Gomez-Sanchez, 2000) this single nucleotide overhang can be used in TA cloning

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where the vector contains a single T over-hang which allows hybridisation of the product into the vector.

Ligation was carried out using a Takara DNA ligation kit. To 2 µl DNA 1 µl of pGEM-T Easy plasmid and 3 µl of the Takara mighty mix were added on ice. This was then incubated over night at 16 °C. The mighty mix contains a T4 DNA ligase which joins the ends of the plasmid with that of the PCR product.

2.3.3.6 Transformation

The plasmid containing the PCR product is transformed into Invitrogen Max Efficiency® DH5α™ Competent Cells to amplify the plasmid containing the DNA of interest. Blue/white selection was used to identify cells that contain the plasmid with an inserted DNA fragment. The β galactosidase enzyme normally converts its substrate X-gal to a blue precipitate in the presence of an inducer IPTG which results in the formation of blue colonies. When a DNA fragment is inserted into the MCS, the β galactosidase gene is disrupted. When this gene is disrupted the enzyme cannot convert its substrate X-gal into a blue precipitate, instead white colonies are formed.

The same transformation protocol was followed as in sub cloning, section 2.3.2.6 above, except blue/white selection could be used with the PGem-Teasy plasmid. When the LB/Amp plates were set 40 µl of 20 mg/ml IPTG and 20 µl of 20 mg/ml X-gal was spread onto each plate as they are the substrates for blue/white selection. After an hour the transformed bacteria were spread onto the plates and left agar-up at 37 °C over night. The following morning blue and white colonies of E. coli could be seen growing on the plates. They were left in the fridge until ready for picking for the minipreps.

2.3.3.7 Minipreps

Minipreps are carried out to isolate the amplified plasmid from the bacterial culture. Usually only 6 minipreps were needed as TA cloning usually has a high efficiency and blue/ white selection was used. 6 white colonies were picked and

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the same protocol was followed as in section 2.3.2.7 to give purified plasmid DNA containing the insert of the gene of interest.

2.3.3.8 Restriction Analysis for Insert

To see if the miniprep clones contained the gene of interest 3 µl of the purified DNA was analysed by restriction digest with EcoR1 and the DNA was digested over night at 37 °C. There are Eco R1 sites on either side of the MCS of P-Gem, so the product obtained should be the same size as the product produced from the primers for the gene of interest.

2.3.3.9 Restriction Analysis for Orientation

Restriction digest was then carried out with the clones that had the correct sized insert to determine the orientation of the PCR product. As TA cloning was used the PCR fragment could be inserted in the forward or reverse orientation. The genes of interest were analysed on Vector NTI to determine what restriction enzymes can be used to produce two fragments of different size to determine what orientation the genes are in. The restriction site should be present in the PCR fragment and in the MCS to get appropriate sized products.

2.3.3.10 Glycerol Stocks

One clone showing the correct restriction analysis for each gene was used to make a glycerol stock. 850 µl of the bacterial suspension saved from the miniprep was added to 150 µl of pure glycerol in a cryovial. The stocks were flask frozen by immersion in liquid nitrogen for a few seconds, and then stored at - 80°C until required.

2.3.3.11 Maxi Preparation of DNA

A maxi prep was carried out to get enough DNA for sequencing and for probe template preparation. The same procedure was followed as described in 2.3.2.9. The DNA was pellet was dissolved in T.1E or DEPC water, concentration determined on the Nanodrop and sent for sequencing.

48 2.3.3.12 Sequencing

An aliquot of maxiprep DNA was sent to the Waikato University DNA sequencing facility for sequencing. This is to ensure the insert is in fact the correct one and that the ligation sites are correct. Primers were supplied by the sequencing facility and used either SP6 (reverse orientation) or T7 (forward orientation) polymerases. The sequence of the insert can then be compared to that of the original by using vector NTi software. Any mismatches can be checked if they are real by looking at the chromatogram to see if an incorrect call has been made by the software.

2.3.3.13 Probe Template Preparation

To prepare a RNA probe the template must be linearised first by restriction enzymes. Restriction enzymes to use were determined by finding an enzyme cuts uniquely on the 5’ end and that creates a 5’ overhang. If the insert was in the forward orientation in the plasmid then the SP6 RNA polymerase was used to create an antisense RNA copy of the DNA. And if in the opposite orientation the T7 RNA polymerase was used.

10 µg of the gene of interest DNA was digested at 37 °C for 2 hrs. After 2 hr 1.25 µl of 2 mg/ml Proteinase K was added and incubated at 37 °C for a further 30 min. RNase free solutions were used from here on. After 30 min, 50 µl of TE was added and a phenol chloroform extraction was performed with through vortexing and centrifugation for 2 min at 13 K. The top aqueous layer was transferred into a new tube. Chloroform extraction was performed by adding 100 µl of pure chloroform to the supernatant tube followed by vortexing and centrifuged again. The top aqueous layer was transferred to a new tube and 10 µl of 3 M sodium acetate (pH 5) and 250 µl of 100 % ethanol was added. The solution was mixed and left at -20 °C for at least 3 hr. After this time the samples were centrifuged at 4 °C for 15 min and the pellet was washed with 70 % ethanol, centrifuged again, the ethanol was aspirated and the pellet was air dried. The pellet was dissolved in 15 µl RNase free 10 mM tris, 0.1 mM EDTA. The concentration was estimated by running a 1:10 dilution of the DNA on a 1 % gel against 2 and 5 µl of a lambda standard.

49 2.3.3.14 Preparation of Riboprobes

Dig probes were prepared from the following mixture:

10 x transcription buffer 2 µl

10 x Dig labelling mix (Boehringer) 2 µl

RNase Out (invitrogen) 1 µl

RNA polymerase (T7 or SP6) (Boehringer) 2 µl

Linearised DNA template 1 µg

DEPC to 20 µl

The reaction mix was left a 37 °C for 2-3 hrs to allow the RNA polymerase produce an RNA copy of the DNA. After 2-3 hrs the RNA was run on a 1.4 % gel with a Lambda DNA ladder. A thin sharp slow migrating DNA band was expected to be seen along with two intense RNA bands that were smaller in molecular weight. If bands were what was expected they were run through a Roche quick spin column for RNA. This column works by size exclusion, where the larger molecules will be eluted at an earlier time than the smaller molecules as they get absorbed into the matrix. This allows the separation of nucleic acids from other molecules like the labelling mix. The probe was then ethanol precipitated by adding 2 µl of 4 M lithium chloride and 3 volumes of ethanol and incubated overnight at -20 °C. After this time the RNA was centrifuged at 13,000 rpm for 15 min, washed with 75 % ethanol and air dried. The pellet was resuspended in 20 µl of DEPC water. The RNA concentration of the probe was estimated by using the nanodrop and then run on a 1.4 % agarose gel.